Combination reader

ABSTRACT

An apparatus for imaging an array of a plurality of features associated with a sample tile. The apparatus includes a stage that supports the sample tile in an illumination region, and an illumination source having a plurality of LEDs adapted to emit light. At least a portion of the light illuminates the illumination region. Additionally, the apparatus includes an image collecting device adapted to selectively collect images of either a first signal when the illumination source is illuminating the illumination region, or a second signal absent illumination of the illumination region. The first signal has wavelengths effectively different from the wavelengths of the portion of the light emitted by the LEDs that illuminates the illumination region.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.11/188,243 filed Jul. 22, 2005, which is a divisional of U.S. patentapplication Ser. No. 10/384,995 filed Mar. 10, 2003, now U.S. Pat. No.6,970,240. The disclosures of the above applications are incorporatedherein by reference.

FIELD

The invention relates generally to imaging biomolecular or syntheticarrays.

BACKGROUND

Substrate-bound biomolecular or synthetic arrays, such asoligonucleotide arrays, also known as micro arrays, enable the testingof the hybridization of different sequences in a sample to manydifferent probes. These arrays can be composed of hundreds of thousandsof probes deposited or synthesized within specific regions, defined asfeatures, on a substrate.

To analyze such arrays, the sample is labeled with one or moredetectable markers, such as fluorescent or chemiluminescent makers, thathybridize with the probes at each feature on the substrate. The markersemit luminous signals, for example a fluorescent signal or achemiluminescent signal, that are imaged and the images are analyzed.

SUMMARY

In various configurations, an apparatus is provided for imaging an arrayof a plurality of features associated with a sample tile. The apparatusincludes a stage that supports the sample tile in an illuminationregion, and an illumination source having a plurality of LEDs adapted toemit light. At least a portion of the light illuminates the illuminationregion. Additionally, the apparatus includes an image collecting deviceadapted to selectively collect images of either a first signal when theillumination source is illuminating the illumination region, or a secondsignal absent illumination of the illumination region. The first signalhas wavelengths effectively different from the wavelengths of theportion of the light emitted by the LEDs that illuminates theillumination region.

Also, in various configurations, a method is provided for collectingimages of fluorescent and chemiluminescent signals using an imagingapparatus. The method includes placing a sample tile on a movable stageof the imaging apparatus, wherein the sample tile includes an array offeatures. At least a portion of the features include at least onehybridized fluorescent marker and/or at least one hybridizedchemiluminescent marker. Additionally, the method includes flooding thesample tile with light utilizing an illumination source of the imagingapparatus, thereby exciting the fluorescent marker in the array. Theillumination source may include a plurality of LEDs. Furthermore, themethod includes collecting images of at least a portion of the arrayutilizing an image collecting device of the imaging apparatus. Theimages selectively showing fluorescent signals emitted by thefluorescent markers when the illumination source floods the sample tilewith light, and/or a plurality of chemiluminescent signals emitted bythe chemiluminescent markers absent the light from the illuminationsource.

BRIEF DESCRIPTION OF THE DRAWINGS

The present invention will become more fully understood from thedetailed description and accompanying drawings, wherein;

FIG. 1 is a perspective view of an imaging apparatus for collectingimages of fluorescent and chemiluminescent hybridized markers in abiomolecular or synthetic sample;

FIG. 2 is a perspective view of an illuminator shown in FIG. 1;

FIG. 3 is schematic of a cross-section of the imaging apparatus shown inFIG. 1, illustrating illumination patterns of the illuminator shown inFIG. 2;

FIG. 4 is a schematic of a cross-section of the imaging apparatus shownin FIG. 1, illustrating the path of the chemiluminescent signals emittedfrom an array of features; and

FIG. 5 is a flow chart for the basic operation of the imaging apparatusshown in FIG. 1.

DETAILED DESCRIPTION

The following description is merely exemplary in nature and in no wayintended to limit the invention, its application, or use.

FIG. 1 is a perspective view representative of various configurations ofan imaging apparatus 10 for collecting images of fluorescent andchemiluminescent hybridized markers in a biomolecular or syntheticsample. The imaging apparatus 10 includes a base 14, a frame 18connected to the base 14, and a mid-support 22 coupled to the frame 18.Additionally, the imaging apparatus 10 includes a transport 26 and anelevator 30 that are controlled by a controller (not shown) to orient astage 34 under an illuminator 38 that illuminates a sample tile 42positioned on the stage 34. The sample tile 42 is a support, such asglass, ceramic, or plastic, to which at least one feature of a sample(not shown) is associated, i.e. placed, synthesized, or attached. Thefeature can be, for example, any feature of the sample where afluorescent and/or chemiluminescent marker has hybridized with a probeattached to the sample tile 42. For example, the feature can be aco-spotted oligonucleotides labeled with one fluorescent marker and onechemiluminescent marker.

In various configurations, the sample tile 42 includes an array ofassociated features having, for example, hundreds or thousands offeatures. In some configurations, the sample tile 42 includes amicroarray having a larger plurality of associated features, forexample, tens of thousands or hundreds of thousands of features. For thesake of convenience and clarity, exemplary configurations will bedescribed below referencing an array of features, but it will beunderstood that the array could include as few as one feature, or thearray could include as many as hundreds of thousands of features, ormore.

In various configurations the array of features is a nucleic acidmicroarray. Such microarrays are becoming an increasingly important toolin bioanalysis and related fields. Nucleic acid microarrays have beendeveloped and find use in a variety of applications, such as genesequencing, monitoring gene expression, gene mapping, bacterialidentification, drug discovery, and combinatorial chemistry. One area inparticular in which microarrays find use is in gene expression analysis.Current methods of manufacturing nucleic acid microarrays, and methodsof their use is diagnostic assays have been described in U.S. Pat. Nos.6,413,722, 6,215,894, 6,040,193, 6,040,138, and 6,387,675.

Furthermore, the imaging apparatus 10 in various configurations includesa first lens 46, a second lens 50, a first filter 54, and an imagecollecting device 58. The first and second lenses 46 and 50 can be anylenses suitable for optical imaging performance, for example mediumformat photographic lenses. In some configurations not illustrated, asingle lens is used for optical imaging performance. In variousconfigurations, the first filter 54 is a longpass filter adapted to passlight having longer wavelengths, for example light having a wavelengthgreater than about 670 nm, or the first filter 54 is a bandpass filteradapted to pass light having wavelengths included in a certain range ofwavelengths, for example light having wavelengths that are between about670 nm and about 700 nm.

The image collecting device 58 and the second lens 50 are positioned inrelation to each other such that a primary imaging surface 62 of imagecollection device 58 is at the focal plane of the second lens 50. Thecontroller utilizes the transport 26 and the elevator 30 to position thestage 34 such that the tile 42 is at a focal plane of the first lens 46.The transport 26 moves the stage 34 along an x-axis, while the elevator30 moves the stage 34 along a z-axis. Both the transport 26 and theelevator 30 are controlled by software via the controller, whichinterfaces with a computer workstation (not shown). Through theworkstation, a user enters a command, e.g. “load sample”, which iscommunicated to the controller. The controller interprets the commandand utilizes at least one motor (not shown) to move the stage along thex-axis and z-axis to the commanded position. In various configurationsthe workstation is separate from the imaging apparatus 10. In othervarious configurations the imaging apparatus 10 includes theworkstation. In other various configurations, the imaging apparatus 10includes various computer workstation components, such as memory and aprocessor, while other computer workstations components, such as agraphical user interface, are separate from the imaging apparatus 10.

In various configurations, the controller and the transport 26 move thestage 34 to pre-set x-axis positions when loading the tile 42 andimaging the features of the array. For example, in some configurations,the controller is configured to instruct the transport 26 to move thestage 34 to a “loading the sample” position, an “imaging position #1”under illuminator 38, and an “imaging position #2” under the illuminator38. The elevator 34 is controlled by the controller to position thestage 34 at the focal plane of the first lens 46. The elevator 30 movesthe stage 34 along a z-axis, while the first and second lenses 46 and 50remain stationary to achieve an optimum focus of the array for the imagecollecting device 58. An algorithm processes image data collected byimage collecting device 58 to determine the position for optimum focusof the array. Therefore, an image of the array is auto-focused for theimage collecting device 58 without adjusting the first and second lenses46 and 50.

For example, image collecting device 58 collects imaging data andcommunicates the data to the workstation where the algorithm determinesthe clarity of the image. That is, the algorithm analyzes the contrastof the image. If the image does not have a desired contrast, thealgorithm instructs the controller to adjust the position of the stagealong the z-axis. Then another image is collected and the data iscommunicated to the workstation where the algorithm again analyzes thecontrast. This process is repeated until the contrast is maximized, i.e.an optimum focus is achieved. In various configurations, the fluorescentsignals emitted by each fluorescent marker are used by the algorithm toauto-focus the array. In some configurations, the elevator 30 is adaptedto rotate the stage 34 in the x-y plane, and the transport 26 is adaptedto move the stage 34 along the y-axis.

When the stage 34 is positioned under the illuminator 38, at the focalplane of the first lens 46, the image collecting device 58 collects atleast one image of the array of features associated with the tile 42.For example, if the sample tile 42 is in an environment illuminatedusing the illuminator 38, the image collecting device 58 collectsillumination data relating to the intensity of light emitted by thefluorescent marker in each feature. Or, for example, if the sample tile42 is an environment absent light that will interfere with thechemiluminescent signals, the image collecting device 58 collectsillumination data relating to the intensity of light emitted by thechemiluminescent markers in each featurer. The image collecting device58 can be any device suitable for collecting image data emitted from thearray of features. For example, in some configurations, image collectingdevice 58 is configured to be a CMOS detector array. In someconfigurations the image collecting device 58 comprises a charge-coupleddevice (CCD).

FIG. 2 is a perspective view representative of various configurations ofthe illuminator 38 (shown in FIG. 1). The illuminator 38 comprises alight source configured to excite the fluorescent marker in each featureby flooding the entire tile 42 (shown in FIG. 1) with light. That is,the illuminator 38 distributes light over the entire tile 42, excitingthe fluorescent markers in all features associated with the tile 42 atthe same time. Additionally, the illuminator 38 substantially evenlydistributes light over the tile 42, such that approximately the sameamount of light is distributed over the entire tile 42. The evenlydistributed flood illumination provides approximately 360° of light toeach feature, thereby allowing more accurate evaluation of the featureby exciting a greater percentage of the fluorescence of each feature,possibly the entire fluorescence of each feature. More specifically,artifacts, i.e. irregularities, in the top surface are less likely toblock the excitation light from reaching all areas of the top surface ofeach feature. Furthermore, flooding the tile and associated array withlight from approximately 360° allows a shape and a size of each featurein the array to be easily determined.

In various configurations, the illuminator 38 includes an opening 66configured to allow images, i.e. fluorescent and/or chemiluminescentlight signals, emitted from each feature to pass through the opening 66.The signals are then re-imaged by the first and second lenses 46, 50(shown in FIG. 1), filtered by the first filter 54 (shown in FIG. 1),and collected by the image collecting device 58 (shown in FIG. 1).Although the illuminator 38 and opening 66 are shown in FIG. 2 as havinga rectangular shape, the illuminator 38 and opening 66, in variousconfigurations, can have any geometric shape suitable to floodilluminate the tile 42. In various configurations, for example, theshape of the illuminator 38 matches the shape of the tile 42. Forexample, in configurations in which the tile 42 is rectangular, theilluminator 38 and opening 66 are also rectangular. In configurations inwhich the tile 42 is round, the illuminator 38 and opening 66 arelikewise round.

Additionally, in various configurations, illuminator 38 can have acontinuous ring form, comprising a single continuous body 64 thatprovides the opening 66, as shown in FIG. 2. Additionally, In variousconfigurations illuminator 38 can have a discontinuous ring form havinga plurality of disconnected sections (not shown) that provides theopening 66. For example, illuminator 38 could have discontinuous ringform comprising two disconnected essentially semi-circular sections, orfour disconnected straight sections that form a rectangular ringdisconnected at the corners.

In various configurations, the illuminator 38 includes a plurality ofLEDs 70, wherein each LED 70 is associated with one of a pluralitysecond filters 74 and one of a plurality of diffusers 78. Forconvenience, the second filters 74 and diffusers 78 are shown in FIG. 2as having different sizes, but are not required to be of different sizesto practice the invention. In some configurations, second filters 74 anddiffusers 78 have the same size and same geometric shape, but in someconfigurations, the second filters 74 and diffusers 78 have differentsizes and geometric shapes. Each LED 70 is enclosed in one of aplurality of recesses 82 that are covered by second filters 74 anddiffusers 78. However, in some configurations, illuminator 38 includes aplurality of any suitable excitation light sources other than LEDs 70,for example, tungsten or xenon bulbs, a laser light source, and/or afiber optic light source.

The LEDs 70 are configured to emit a wavelength of light at an intensitylevel that excites a fluorescent marker in each feature. For example, insome configurations, the illuminator 38 includes LEDs 70 that emit lighthaving a wavelength of about 635 nm to excite fluorescent markers thatemit red light. In some configurations, the illuminator 38 includes LEDs70 that emit light having a wavelength of about 470 nm used to excitefluorescent markers that emit blue light. Other wavelengths may be usedto excite fluorescent markers having other excitation requirements. Invarious configurations the Illuminator 38 includes LEDs 70 that emitlight having various wavelengths. For example, various LEDs 70 emitlight having a wavelength of 635 nm, while other LEDs 70 in illuminator38 emit light having a wavelength of 470 nm, and other LEDs 70 may emitlight having other wavelengths. This would allow the use of multi-colorfluorescent markers in the array of features.

In various configurations, imaging apparatus 10 is configured to allowthe illuminator 38 to be removed and replaced with an illuminator 38comprising LEDs that emit light having a different wavelength. Thus, iftile 42 associated with an array of features having fluorescent markersthat emit red light is removed and replaced with a tile 42 associatedwith an array of features having fluorescent markers that emit bluelight, the illuminator 38 can be removed and replaced accordingly.

Furthermore, in some configurations, each of the LEDs 70 is oriented inthe recesses 82 so that light provided by each LED 70 is directed towardone or more desired areas of the tile 42. For example, each LED 70 canbe oriented so that light emitted from each LED is generally directed tothe center of the tile 42, or each LED 70 can be oriented so that lightemitted from each LED is directed to different sections of the tile 42.In various configurations, a front face 84 of the illuminator 38 isangled inward to allow the LEDs 70 to point downward and slightly inwardtoward a focal point in the center of the tile 42.

In some configurations, the diffusers 78 diffuse light emitted from eachLED 70 to substantially evenly distribute the light from each LED 70over the entire tile 42. That is, diffusers 78 have a divergence angleselected so that light emitted from each LED 70 illuminates the entiretile 42. Therefore, the light emitted from each LED 70 overlaps with thelight emitted from each of the other LEDs 70. Thus, the intensity oflight provided by the illuminator 38, over the entire tile 42 is afunction of the number of LEDs included in the illuminator 38 and theselected intensity of the LEDs 70. In some configurations, a singlediffuser (not shown) is used. In various configurations the singlediffuser has the same shape as the front face 84 of illuminator 38. Thesingle diffuser covers each LED 70 and simultaneously diffuses the lightemitted from each LED 70. In various other configurations at least twodiffusers (not shown) are used to diffuse light emitted by the LEDs 70.

The second filters 74 eliminate light emitted by the LEDs 70 having awavelength that would reflect off the array, the tile 42, or the stage38 and undesirably pass through the first filter 54 to the imagecollecting device 58. For example, in some configurations, the firstfilter 54 passes light having a wavelength greater than about 640 nm,and the second filter 74 passes only light having a wavelength of lessthan about 635 nm. In some configurations, the second filters 74 areshortpass filters adapted to pass light having shorter wavelengths, forexample light having a wavelength less than about 635 nm. In someconfigurations, the second filter 74 is a bandpass filter adapted topass light having wavelengths included in a certain range ofwavelengths, for example light having wavelengths that are between about550 nm and about 635 nm. In various configurations, the apparatus 10includes a single second filter (not shown for eliminating light emittedby the LEDs 70. In various other configurations, the apparatus 10includes two or more second filters (not shown), whereby each of thesecond filters 74 filters light emitted by at least one of the LEDs 70.

In various configurations, EPI illumination is utilized, in place of theilluminator 38, to illuminate the array and excite the fluorescentmarkers. An EPI based system would have a dichroic beam splitter (notshown) between the first lens 46 and the first filter 54. Light emittedfrom the EPI illuminator would be shaped and imaged onto the sample tile42 through the first lens 46. Leds, a lamp or a laser could be used asthe illumination source. Any suitable illumination source can beutilized to illuminate the array and excite the fluorescent markers. Forexample, off axis illumination and electro luminescent panels can beutilized.

Referring to FIG. 1, the first filter 54 is positioned between the firstlens 46 and the second lens 50 when it is desirable to filter out lightreflecting off the array from the illuminator 38 having certainwavelengths. Therefore, light emitted from the illuminator 38 thatoverlaps with the fluorescent emissions of the array of features isseparated from the fluorescent emissions and substantially preventedfrom reaching the image collecting device 58. The first filter 54 can beremoved when filtering is not desired, for example, whenchemiluminescent emissions are to be imaged all light the can interferewith the enzymatically generated chemiluminescent signals must besubstantially removed from the environment surrounding the imagingapparatus 10. In some configurations, the removal and insertion of thefirst filter 54 is automated by the controller and a mechanism (notshown) suitable for inserting the first filter 54 between the first andsecond lenses 46 and 50, and removing the first filter 46 when desired.

In some configurations, a filter wheel having a plurality of filters isused as a first filter 54, wherein each filter of the filter wheelfilters out light of a different wavelength, or within a differentbandwidth. Positioning of the filter wheel is automated by thecontroller and a mechanism suitable to rotate the wheel such that adesired filter, or no filter, is positioned between the first and secondlenses 46 and 50. The first filter 54 works in combination with thesecond filter 78 to allow only fluorescent emissions of the array to becollected by the image collecting device 58 when the illuminator 38 isilluminated.

FIG. 3 is a schematic of a cross-section of various configurations ofthe imaging apparatus 10 (shown in FIG. 1), illustrating the floodillumination of the illuminator 38 (shown in FIG. 2) and the path of thefluorescent signals emitted from an array of features. Each LED 70 emitslight directed at the tile 42 and the associated array. The lightemitted by each LED 70 is filtered by the second filter 78 so that onlylight having a desired wavelength, or within a desired range ofwavelengths, illuminates the tile 42 and associated array. Additionally,light emitted from each LED 70 is diffused by the diffuser 78 to providea substantially uniform intensity of light over the entire tile 42, asindicated by LED illumination pattern lines 86. Therefore, the lightemitted from each LED 70 overlaps with the light emitted from at leastone of the other LEDs 70, as generally indicated at overlap area 90.

The light emitted by the LEDs 70, filtered by the second filters 74, anddiffused by the diffusers 78, excites the fluorescent markers in eachfeature of the array, resulting the emission of fluorescent signals 94.The fluorescent signals 94 pass through the opening 66 in theilluminator 38 and enter the first lens 46, where they are re-imaged.The signals 94 are then filtered by the first filter 54, which filtersout any light from the LEDs 70 that has reflected off of the array offeatures, the tile 42 and/or the stage 34. The filtered signals 98 thenpass through the second lens 50 where they are re-imaged again. Afterpassing through the second lens 50, the fluorescent signals 94 arecollected by image collecting device 58, and the collected image data istransmitted to a computer based system (not shown), where the data isprocessed and analyzed.

FIG. 4 is a schematic of a cross-section representative of variousconfigurations of imaging apparatus 10 (shown in FIG. 1), illustratingthe path of the chemiluminescent signals emitted from an array offeatures. To collect images of chemiluminescent signals 102 emitted bythe feature in the array, the first filter 54 (shown in FIG. 3) isremoved from between the first and second lenses 46 and 50, and theilluminator 38 is turned off. The chemiluminescent signals must beimaged in a substantially light free environment. That is, anenvironment substantially free from any light that will interfere withthe chemiluminescent signals emitted from the array.

In various configurations the chemiluminescent signals are enzymaticallygenerated. Methods for generating chemiluminescent signal inbiomolecular array, for example nucleic acid microarrays, have beendescribed in U.S. Pat. Nos. 5,625,077, 5,652,345, 5,679,803, 5,783,381,6,022,964, 6,133,459, and 6,124,478.

The chemiluminescent signals 102 emitted from the array pass through thefirst and second lenses 46 and 50, where the chemiluminescent signals102 are re-imaged by each lenses 46 and 50. After passing through thelenses 46 and 50, the chemiluminescent signals 102 are collected byimage collecting device 58. The collected image data is then transmittedto the computer based system, where the data is processed and analyzed.In various configurations, each feature may have more than onechemiluminescent marker hybridized with probes associated with the tile42. In which case, the first filter 54 would not be removed in order tofilter out light emitted from one of the chemiluminescent markers of thefeatures while allowing wavelengths of different chemiluminescentsignals to pass and be imaged by the image collecting device 58. Thefirst filter 54 would then be removed and replaced with a differentfirst filter 54 that would allow other chemiluminescent signals to beimaged.

Referring now to both FIGS. 3 and 4, in various configurations, thefiltered fluorescent signals 98 collected by image collecting device 58are used to auto-focus the array of features for the image collectingdevice 58, as described above in reference to FIG. 1, for examplecorrections for chromatic aberrations are made. Additionally, thefiltered fluorescent signals 98 collected by image collecting device 58are used for gridding the array of features. That is, the filteredfluorescent signals 98 are used to identify the location of each featurewithin the array. Furthermore, the filtered fluorescent signals 98collected by image collecting device 58 are used to normalize the array.More specifically, the filtered fluorescent signals 98 are used tonormalize the chemiluminescent signals 102 collected by the imagecollecting device 58.

FIG. 5 is a flow chart 200 representative of various methodconfigurations for operating an imaging apparatus 10 for imaging anarray of features. To begin, a user positions the tile 42 and associatedarray of features onto the stage 34, as indicated at 202. The controllerinstructs the transport 26 to move the stage 34 along the x-axis to afirst position under the illuminator 38, where the array is illuminatedby the illuminator 38, as indicated at 204. Next the first filter 54 ispositioned between the first and second lenses 46 and 50, as indicatedat 206. The array is then auto-focused for the image collecting device58 by moving the stage 34 along the z-axis, via the elevator 30, asindicated at 208. A normalizing image of the fluorescent signals 98emitted by each feature in a first portion of the array is thencollected, as indicated at 210. Next, the first filter 54 is removedfrom between the first and second lenses 46 and 50, and the illuminator38 is turned off, as indicated at 212, thereby providing a substantiallylight free environment for imaging the chemiluminescent signals emittedby each feature. Then an image of the chemiluminescent signals 102emitted by each feature in the first portion of the array is collectedby the image collecting device 58, as indicated at 214.

Next, in various configurations, depending on the size of the array, thestage 30 is moved to a second position under the illuminator 38, firstfilter 54 is re-positioned between the lenses 46 and 50, and theilluminator 38 is turned on, as indicated at 216. Then, a secondauto-focus procedure is performed, a normalizing fluorescent image of asecond portion of the array is collected, the first filter 54 is againremoved from between the first and second lenses 46 and 50, andilluminator 38 is again turned off, as indicated at 218. An image of thechemiluminescent signals 102 emitted by each feature in the secondportion of the array is then collected by the imaging device 58, asindicated at 220. This process is repeated, as needed, until images ofthe chemiluminescent signals 102 for the entire array have beencollected, as indicated at 222.

Thus, the imaging apparatus of the present invention automaticallyacquires multiple images of an array of fluorescent/chemiluminescentco-hybridized features, thereby acquiring image data for the entirearray using a single apparatus. Additionally, the present inventionallows better alignment between the fluorescent and the chemiluminescentimage data because the optics are the same for the collection in bothchannels. Furthermore, the illuminator substantially evenly distributesexcitation light over the entire array, thereby providing moreconsistent image data for multiple images across the entire array.

While the invention has been described in terms of variousconfigurations, those skilled in the art will recognize that theinvention can be practiced with modification within the spirit and scopeof the claims.

1. An illumination source for illuminating an array including aplurality of features to be imaged by an imaging system, theillumination source comprising; a frame configured to be coupled to theimaging system; a plurality of LEDs associated with the frame, the LEDsconfigured to emit light directed at the array, thereby resulting in afluorescent signal being emitted by at least one feature in the array;and a plurality of diffusers configured to diffuse the light emittedfrom each LED such that the light emitted from each LED overlaps thelight emitted from at least one other LED at a top surface of the array,thereby flooding the array with light from a plurality of directions sothat at least one of a shape and a size of at least one feature in thearray is determinable.
 2. The illumination source of claim 1 furthercomprising a plurality of filters, wherein each filter is associatedwith one of the LEDs, and each filter is configured to filter the lightemitted by the corresponding LED such that light, other than lighthaving a wavelength suitable to result in the emission of thefluorescent signal, is effectively prevented from passing through eachsecond filter.
 3. The illumination source of claim 1, wherein theillumination source comprises an illumination ring having a singlecontinuous body that forms a ring with an opening therethrough.